CN114163420A

CN114163420A - Endoplasmic reticulum Golgi matrix targeting small molecule, conjugate and application thereof

Info

Publication number: CN114163420A
Application number: CN202111251211.9A
Authority: CN
Inventors: 王骥; 王夏峰; 黄章屏; 尚丽茹; 蒋娟; 邓蔡冠曦
Original assignee: First Affiliated Hospital of Sun Yat Sen University
Current assignee: First Affiliated Hospital of Sun Yat Sen University
Priority date: 2021-10-26
Filing date: 2021-10-26
Publication date: 2022-03-11
Anticipated expiration: 2041-10-26
Also published as: CN114163420B

Abstract

The invention discloses an endoplasmic reticulum Golgi matrix targeting small molecule, and the structural formula of the small molecule is shown as a formula I. The invention also provides an endoplasmic reticulum Golgi matrix targeted small molecule conjugate, which has a structural formula shown in formula III. The invention also provides application of the small molecule and the conjugate for targeting the endoplasmic reticulum Golgi to preparation of a STING protein agonist, or for improving the antigen cross presentation level, or for improving and enhancing the antigen targeting endoplasmic reticulum Golgi, or for preparing a medicament for inducing CD8+ T immune response, or for preparing an immunotherapy medicament. The endoplasmic reticulum Golgi targeted small molecule and the conjugate provided by the invention can realize good connection between the endoplasmic reticulum Golgi targeted small molecule and an antigen, maintain the binding force of a bis-benzopyrimidine skeleton to STING protein, reduce systemic inflammatory reaction caused by over-diffusion of STING agonist DiabZI, enhance the targeting of the antigen to the endoplasmic reticulum Golgi at a subcellular level, improve the cross presentation level of the antigen, promote the maturation of DC cells and efficiently induce CD8+ T immune reaction.

Description

Endoplasmic reticulum Golgi matrix targeting small molecule, conjugate and application thereof

Technical Field

The invention belongs to the field of biomolecule chemistry, and particularly relates to endoplasmic reticulum Golgi matrix targeted small molecules, a conjugate and application thereof.

Background

Induction of CD8+ T cell responses is critical to the response of viral prophylactic vaccines to viral variation and is central to tumor therapeutic vaccines. In order to improve the CD8+ T cell reaction, the improvement of the cross presentation of antigen presenting cells (especially dendritic cells) is key, but under natural conditions, the antigen presentation of dendritic cells is influenced by a plurality of factors such as the efficiency of antigen entering the cells, the sub-localization of antigen in the cells, the activation degree of the dendritic cells and the like, so that the low efficiency is shown.

In order to improve the antigen presentation efficiency of dendritic cells, two aspects need to be started: 1) maturation status of DC cells; 2) antigen delivery into the interior of the cell to form the antigen-major histocompatibility complex I (pMHC-I). Because antigens often exhibit poor immunogenicity, additional immunoadjuvants are required to promote DC cell maturation, and are currently administered in most cases by mixed injection of the immunoadjuvant and the antigen, with the adjuvants typically freund's adjuvant, aluminum hydroxide adjuvant, or natural ligands or synthetic agonists of Pattern Recognition Receptors (PRRs) that predispose to B cell responses. After the antigen enters the dendritic cell, the main delivery route is the cytosolic route, i.e. the antigen and major histocompatibility complex assemble in the endoplasmic reticulum and further optimize the final presentation to the cell surface in the golgi apparatus. It was found that optimization of the cytoplasmic pathway by targeted delivery to the er-golgi matrix is an effective means of elevating the number of pMHC-I. At present, the method is mainly realized by adding polypeptide or signal peptide which targets endoplasmic reticulum Golgi matrix at the N end of the antigen, and less research is carried out on non-polypeptide molecules.

The immune adjuvant is adopted for mixed injection administration, so that the systemic diffusion of the immune adjuvant is easily caused, the effect of inducing dendritic cells is reduced, and the systemic inflammatory reaction is initiated. If the targeting polypeptide is used, the endoplasmic reticulum targeting polypeptide is used as a foreign antigen to easily induce an organism to generate immune reactions aiming at the targeting polypeptide, and the immune reactions can remove the whole vaccine together when the targeting polypeptide is used for the second time, so that the same system cannot be used for multiple times.

Interferon gene stimulating factor (STING), a transmembrane protein located on the endoplasmic reticulum of subcellular structures, is triggered by cytoplasmic DNA from pathogens and hosts as a key signal transduction molecule involved in innate immune responses, and plays an important role in inducing secretion of type I interferons and proinflammatory cytokines, defending against viral and intracellular bacterial infections, and regulating the development of spontaneous antitumor immune responses in vivo. Many studies have shown that STING is involved in the development of a variety of diseases, and that STING activation can induce an effective immune response against pathogenic infections and cancer; abnormal activation of STING can lead to autoimmune and inflammatory diseases. As a potential new target for drugs, STING has also been extensively studied for its structure and function. STING agonists are effective in the treatment of pathogen infections and cancer. The study of STING and its agonists has progressed rapidly over the last 10 years. In 2018, the Kuransu Schk company discovers a class of aminobenzimidazole compound dimers (DiabZI) through high-throughput screening, and designs the aminobenzimidazole compound into the dimers through analysis of a protein cocrystal structure, so that the aminobenzimidazole compound can be respectively combined with a STING dimer, and the activity is improved by 1000 times. The compound DiaBZI can effectively inhibit the growth of tumors, keep the concentration in the tumors higher than that in blood, and simultaneously can generate release of cytokines such as IFN-beta and the like higher than those in blood plasma in the tumors, thereby realizing systemic drug delivery.

After being activated, the STING protein can be transported to the Golgi apparatus, and the transport route is highly consistent with the cross presentation of the antigen, so that the antigen can be delivered at the subcellular level by means of the high affinity of the Diabzi to the STING protein, the concentration of the antigen in endoplasmic reticulum and Golgi apparatus is increased, and the number of antigen-major histocompatibility complexes is increased. Furthermore, DiabZI can activate cGAS-STING pathway to induce cytokine production such as interferon-type I and promote maturation of DC cells. Through both elevations, high levels of CD8+ T cell responses are ultimately induced.

Disclosure of Invention

The invention aims to solve the technical problems and provides a technical scheme which can effectively combine the STING protein, enhance the antigen to target endoplasmic reticulum and Golgi apparatus at a subcellular level and improve the antigen cross presentation level.

In order to achieve the above object, the present invention provides an endoplasmic reticulum golgi targeted small molecule, the structural formula of which is shown in the following formula (I):

on the other hand, the invention also provides application of the endoplasmic reticulum Golgi targeting small molecule in preparing a STING protein agonist.

On the other hand, the invention also provides application of the endoplasmic reticulum Golgi targeted small molecule in preparing a medicament for improving the antigen cross presentation level.

On the other hand, the invention also provides application of the endoplasmic reticulum Golgi targeting small molecule in preparing a medicament for enhancing antigen targeting to the endoplasmic reticulum.

On the other hand, the invention also provides application of the endoplasmic reticulum Golgi targeting small molecule in preparing a medicament for inducing CD8+ T immune response.

On the other hand, the invention also provides application of the endoplasmic reticulum Golgi targeting small molecule in preparing an immunotherapy medicament.

In addition, the invention modifies a dipyridamole STING protein agonist (DiabZI), extends a hydrophilic region of a binding pocket thereof, and covalently connects antigens (including polypeptides and proteins) on the premise of not influencing the binding force of the DiabZI to form an endoplasmic reticulum Golgi matrix targeted conjugate, wherein the structural formula of the conjugate is shown as the following formula (II):

wherein R is₁Is that

R₂Is that

R₃Is an antigen.

Preferably, the antigen is a polypeptide antigen or a protein antigen, in particular an antigen containing a cysteine at the N-terminus.

Preferably, R₃And R₂The connection mode is as follows:

in another aspect, the invention also provides the application of the Neurone Golgi targeted conjugate in preparing a STING protein agonist.

On the other hand, the invention also provides application of the endoplasmic reticulum Golgi targeting conjugate in preparing a medicament for improving the antigen cross presentation level.

On the other hand, the invention also provides application of the endoplasmic reticulum Golgi targeting conjugate in preparing a medicament for enhancing antigen targeting to the endoplasmic reticulum.

On the other hand, the invention also provides application of the Neurone Golgi targeted conjugate in preparing a medicament for inducing a CD8+ T immune response.

On the other hand, the invention also provides application of the endoplasmic reticulum Golgi targeted conjugate in preparing an immunotherapy medicament.

Therefore, the endoplasmic reticulum Golgi targeted small molecule and the conjugate provided by the invention can realize good connection between the endoplasmic reticulum Golgi targeted small molecule and an antigen, can better keep the binding force of a bis-benzopyrimidine skeleton to a STING protein, can reduce systemic inflammatory reaction caused by over-diffusion of a STING agonist DiabZI, can enhance the targeting of the antigen to the endoplasmic reticulum at a subcellular level, improve the antigen cross presentation level, and can promote the combination of the antigen delivery into cells to form the number of antigen-major histocompatibility complex I (pMHC-I) by promoting the maturation of DC cells, thereby realizing the high-efficiency induction of CD8+ T immune reaction.

The present invention is illustrated with the CD8+ T epitope (CSGLEQLESIINFEKI (SEQ ID NO: 1), COVA-i) of mutant Ovalbumin (OVA) as an exemplary model for validation in bone marrow derived dendritic cells (BMDC). Compared with a mixed group of the DiaBZI and the COVA-i, the coupling of the DiaBZI-COVA-i can obviously improve the cross presentation of the BMDC to the COVA-i and simultaneously show a considerable activation effect. After covalent coupling of OVA-i polypeptide containing FITC fluorophore and diABZI, subcellular level aggregation of endoplasmic reticulum can be realized. Finally, O-T1 mouse-derived CD8+ T cells were co-incubated with the treated BMDCs of COVA-i, and the BMDCs of the DiaBzi-COVA-i coupled group could more efficiently expand CD8+ T cells.

Drawings

FIG. 1 is a UPLC spectrum of DiaBZI (A) and DiaBZI-MAL (B)

FIG. 2 shows the HPLC spectrum (A) and MALDI-TOF spectrum (B) of the conjugate diABZI-COVA-i.

Figure 3 shows that diABZI targeted delivery of antigen COVA-i elevates the level of cross presentation by BMDC.

Fig. 4 shows the targeted delivery of conjugated polypeptides containing FITC fluorophores to the endoplasmic reticulum golgi apparatus by diABZI.

FIG. 5 shows that the amplification effect of the BMDCs treated with DiabZI-COVA-i was significantly enhanced after co-incubation with O-T1 mouse-derived CD8+ T cells.

Detailed Description

The present invention will be further described with reference to the following examples. It should be understood that the following examples are illustrative of the present invention only, and are not intended to limit the scope of the present invention.

In the examples below, all reagents used in the experiments were purchased from reagent companies and were not further processed. All reactions were monitored by a 0.2 mm thick thin layer chromatography silica gel plate and ninhydrin, phosphomolybdic acid developing solution and an ultraviolet analyzer, the silica gel used for the flash column chromatography was 200-mesh silica gel powder, the mass spectrometry data was collected by a quadrupole time-of-flight tandem mass spectrometer, the polypeptide (COVA-i, CSGLEQLESIINFEKI (SEQ ID NO: 1)) was custom made from south kyoto jeopard peptide, the ultra high performance liquid chromatograph from watt company was used for compound analysis, the high performance liquid chromatograph used for preparation was the high performance liquid chromatograph from shimadzu corporation, and the detection wavelengths were 220nm and 321 nm. The gene amplification assay uses a fluorescence quantitative PCR instrument of Saimerfi, and the flow cytometry assay uses a flow analyzer of BD, USA. The immunofluorescence observation was performed using an ultra-high resolution confocal laser microscope from zeiss, germany.

Example 1

The synthesis routes shown below were followed to synthesize DiabZI and DiabZI-MAL, respectively.

Reaction and conditions:

I) saturated ammonia water at 25 deg.C for 24 hr, then at 50 deg.C for 4 hr;

II) (4-Aminobut-2-en-1-yl) carbamic acid tert-butyl ester, N, N-Diisopropylethylamine (DIPEA), Dimethylformamide (DMF), 120 ℃, 16h, then trifluoroacetic acid (TFA), Dichloromethane (DCM), 25 ℃, 2 h;

III) boron bromide, DCM at 25 ℃ for 16 h;

IV)4 a: n- (3-chloropropyl) morpholine, DIPEA, DMF, 70 ℃, 16h or 4 b: 4- (3- (tosyloxy) propyl) piperazine-1-carboxylic acid tert-butyl ester, DIPEA, DMF, 80 ℃, 4 h;

v) DIPEA, Isopropanol (IPA) at 120 ℃ for 16-24 h;

VI) sodium dithionite, MeOH and water in a ratio of 4: 3, at 25 ℃ for 15 min;

VII) 1-ethyl-3-methyl-1H-pyrazole-5-carbonyl isothiocyanate, DMF/1, 4-dioxane, 0 ℃ for 30min, then 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDC), DIPEA, 25 ℃ for 6-14H;

VIII) 1-ethyl-3-methyl-1H-pyrazole-5-carbonyl isothiocyanate, DMF/1, 4-dioxane, 0 ℃, 30min, then 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDC), DIPEA, 25 ℃, 6-14H; then TFA, DCM, 25 ℃, 2 h;

IX) succinimidyl 6- (maleimido) hexanoate, DIPEA, DMF, from 0 ℃ to 25 ℃ for 1 h.

1. Preparation of Compound 1

4-chloro-3-methoxy-5-nitrobenzoic acid methyl ester (1g, 4.1mmol) is dispersed in 10ml of saturated ammonia water, stirred for 24 hours at room temperature, heated to 50 ℃ and stirred for 2 hours, supplemented with 2ml of saturated ammonia water, stirred for 2 hours, cooled to room temperature, filtered and washed by ice water, and dried in vacuum to obtain 0.67g of light yellow solid (yield 71%), namely the compound 1.

2. Preparation of Compound 2

Dissolving the compound 1(149mg, 0.65mmol) in 2ml of dry DMF, adding tert-butyl (4-aminobut-2-en-1-yl) carbamate (100mg, 0.54mmol) and DIPEA (0.19ml, 1.1mmol), heating and stirring at 120 ℃ for 16 hours, cooling to room temperature, diluting the reaction solution with ethyl acetate, washing with water, extracting the water phase twice with ethyl acetate, collecting the organic phase, washing with saturated common salt water once, drying with anhydrous sodium sulfate, concentrating, and carrying out column chromatography separation and purification to obtain 98mg of an orange solid.

The product obtained above was dissolved in 2ml of DCM, and 1ml of TFA was added thereto, and stirred at room temperature for 2 hours, followed by concentration and separation and purification by column chromatography to obtain 75mg of brick-red solid (yield of two steps: 50%), i.e., Compound 2.

3. Preparation of Compound 3

Compound 1(500mg, 2.2mmol) was dissolved in 10ml DCM and boron bromide (0.84ml, 8.7mmol) dissolved in 5ml DCM was slowly added to the reaction, stirred at room temperature for 16 hours, added to 50ml ice water, stirred vigorously for 30 minutes, filtered and dried in vacuo to give 299mg of a pale solid (64% yield), Compound 3.

4. Preparation of Compound 4a

Compound 3(500mg, 2.3mmol) is dissolved in 5ml anhydrous DMF and N- (3-chloropropyl) morpholine (0.43ml, 2.8mmol) and DIPEA (0.8ml, 4.6mmol) are added, the mixture is heated and stirred at 70 ℃ for 16 hours, the reaction solution is diluted with ethyl acetate and saturated NaHCO₃The solution was washed, the aqueous phase was extracted twice with ethyl acetate, the organic phase was collected, dried over anhydrous sodium sulfate, concentrated and purified by column chromatography to give 421mg (yield 53%) of a yellow solid, compound 4 a.

5. Preparation of Compound 4b

Compound 3(1g, 4.6mmol) was dissolved in 10ml of anhydrous DMF, tert-butyl 4- (3- (toluenesulfonyloxy) propyl) piperazine-1-carboxylate (1.47g, 3.7mmol) and DlPEA (1.6ml, 9.2mmol) were added, the mixture was stirred at 80 ℃ for 4 hours, the reaction solution was diluted with ethyl acetate and saturated NaHCO₃Washing, extracting the water phase twice with ethyl acetate, collecting the organic phase, drying with anhydrous sodium sulfate, concentrating, and purifying by column chromatography to obtain 733mg (yield 45%) of yellow solid, namely compound 4 b.

6. Preparation of Compound 5a

Compound 4a (421mg, 1.2mmol) is dissolved in 10ml of isopropanol, compound 2(280mg, 1.0mmol) and DIPEA (0.7ml, 4.0mmol) are added, the mixture is heated in a sealed tube at 120 ℃ and stirred for 18 hours, cooled to room temperature, filtered, washed three times with isopropanol and dried in vacuum to obtain 231mg of brick-red solid (yield 39%), namely compound 5 a.

7. Preparation of Compound 5b

Compound 4b (350mg, 0.79mmol) was dissolved in 6ml isopropanol, compound 2(221mg, 0.79mmol) and DIPEA (0.55ml, 3.2mmol) were added, the mixture was stirred at 120 ℃ with sealed tube heating for 21 hours, cooled to room temperature, filtered, washed three times with isopropanol and dried in vacuo to give 236mg of brick red solid (yield 44%) which was compound 5 b.

8. Preparation of Compound 6a

Compound 5a (69mg, 0.12mmol) is dissolved in 2ml of methanol, sodium dithionite (328mg, 90% purity, 1.7mmol) dissolved in 1.5ml of water is added, stirring is carried out at room temperature for 15 minutes, 460mg of NaHCO is added₃The powder was stirred for 10 minutes at room temperature, filtered, washed three times with methanol, and the filtrate was concentrated and purified by column chromatography to give 36mg (yield 58%) of a yellow solid, compound 6 a.

9. Preparation of Compound 6b

Compound 5b (528mg, 0.77mmol) is dissolved in 8ml of methanol, sodium dithionite (2.1mg, 90% purity, 11mmol) dissolved in 6ml of water is added, stirring is carried out at room temperature for 15 minutes, 2.9g NaHCO are added₃The powder was stirred for 10 minutes at room temperature, filtered, washed three times with methanol, and the filtrate was concentrated and purified by column chromatography to give 265mg (yield 55%) of a yellow solid, compound 6 b.

10. Preparation of compound DiabZI

Compound 6a (58mg, 0.11mmol) was dissolved in 2ml of anhydrous DMF and 0.4M solution of 1, 4-dioxane of 1-ethyl-3-methyl-1H-pyrazole-5-carbonyl isothiocyanate (1.1ml, 0.44mmol) was slowly added under ice-bath conditions, after stirring at 0 ℃ for 30 minutes EDC (96mg, 0.50mmol) and DIPEA (153. mu.l, 0.88mmol) were added and slowly warmed to room temperature, stirred for 14 hours, purified by column chromatography, washed with acetonitrile and dried in vacuo to give 29mg (31% yield) of the compound DiabZI. EsI (m/z): [ M + H ]]⁺C₄₂H₅₂N₁₃O₇ ⁺Calculated 850.41, found 850.75.

11. Preparation of Compound 7b

Compound 6b (78mg, 0.12mmol) was dissolved in 2ml of anhydrous DMF and 0.4M solution of 1, 4-dioxane of 1-ethyl-3-methyl-1H-pyrazole-5-carbonyl isothiocyanate (1.2ml, 0.48mmol) was slowly added under ice-bath conditions, after stirring at 0 ℃ for 30 minutes EDC (115mg, 0.60mmol) and DIPEA (167. mu.l, 0.96mmol) were added and slowly warmed to room temperature, stirred for 8 hours, purified by column chromatography and concentrated to give a yellow solid.

The solid was further dissolved in 2ml of DCM, and 1ml of TFA was added thereto, and stirred at room temperature for 2 hours, followed by concentration, separation and purification by column chromatography, and washing with acetonitrile to obtain 12mg of a white solid (yield in two steps, 11%), i.e., Compound 7 b.

12. Preparation of compound DiabZI-MAL

Compound 7b (20mg, 0.024mmol) was dissolved in 1ml anhydrous DMF and succinimidyl 6- (maleimido) hexanoate (9mg, 0.029mmol) and DIPEA (N, N-diisopropylethylamine) (8.4. mu.l, 0.048mmol) were added under ice bath conditions, slowly warmed to room temperature, stirred for 1 hour and purified by column chromatography, washed with acetonitrile and dried under vacuum to give 24mg of a white solid (yield 98%) which was the compound DiabZI-MAL. ESI (m/z): [ M + H ]]⁺C₅₂H₆₄N₁₅O₉ ⁺Calculated 1042.50, found 1042.79.

UPLC (ultra performance liquid chromatography) spectra of dianzi and dianzi-MAL are shown in fig. 1 as a and B, respectively.

The structural formula of the DiaBZI-MAL is shown as the following formula (I):

example 2: preparation of conjugate of DiaBZI-MAL and polypeptide COVA-i

It is to be noted that the present invention herein provides only the preparation of conjugates of diABZI-MAL with the polypeptide COVA-i as an illustrative example, but the antigens referred to herein as being capable of forming conjugates with diABZI-MAL are not limited to such polypeptides and may be any other kind of antigens known in the art. It will be appreciated that those skilled in the art will be able to couple other antigens to the diABZI-MAL in a similar manner to form diABZI-MAL-antigen conjugates.

For cysteine-containing antigens: dissolving the DiabZI derivative in Dimethylformamide (DMF), dissolving the antigen in a weakly alkaline buffer solution, mixing at a molar ratio of 1: 1, incubating at room temperature for 24 hours, separating and purifying by HPLC or FPLC, concentrating, and lyophilizing to obtain powder.

Linked diABZI-antigen conjugate assay: the product was dissolved in a suitable solvent and measured against a standard curve using the absorbance of DiabZI at 321 nm.

The specific synthesis steps are as follows: mixing a 2mmol/L aqueous solution of COVA-i (0.6 mu mmol/300 mu L) with a 2mmol/L solution of diABZI-MAL (0.66 mu mmol/330 mu L) in DMF (dimethylformamide), incubating overnight at room temperature, separating by high performance liquid chromatography, concentrating, and lyophilizing to obtain white powder 0.7mg (yield 41%), which is the conjugate diABZI-COVA-i of the diABZI-MAL and the polypeptide COVA-i. MALDI-TOF (m/z): [ M + H ]]⁺Calculated 2865.426, found 2865.443.

The structural formula of the conjugate DiabZI-COVA-i is shown as follows:

Example 3: DiabZI-COVA-i activation assay and cross-presentation assay

Preparation of BMDC cells (mouse bone marrow-derived dendritic cells)

Tibia bone marrow of 4-6 weeks mice (purchased from the center of the Guangdong province animal) was taken, and dispersed into single cells through a 40 μm cell sieve, and erythrocytes were removed with ACK lysate. Followed by 1X 10⁶Culturing the bone marrow cells in RPMI1640 (containing 10% v/v FBS) containing 20ng/ml granulocyte-macrophage colony stimulating factor (GM-CSF) for 2 days, removing the suspension cells, replacing with new culture medium (RPMI 1640 (containing 10% v/v FBS) containing 20ng/ml granulocyte-macrophage colony stimulating factor (GM-CSF)), continuing to culture for 2 days, removing the suspension cells, replacing with new culture medium (RPMI 1640 (containing 10% v/v FBS) containing 20ng/ml granulocyte-macrophage colony stimulating factor (GM-CSF)), after continuing to culture for 2 days, taking the suspension cells, marking with FITC-CD11c antibody, and separating CD11c + cells by using a flow cytometer to obtain BMDC cells.

Experimental determination of the activation of DiaBZI-COVA-i

The basic operation is as follows: BMDC cells were incubated with different combinations of polypeptides at 37 ℃ and after 4 hours mRNA expression levels of the relevant cytokines IFN-. beta.and CXCL10 were detected by q-PCRThe ability of diABZI and conjugated diABZI to activate STING proteins was evaluated. Selecting BMDC according to 2 × 10⁵The wells were placed in 24-well plates and after 24 hours a final concentration of 1 μ M of COVA-i, a mixture of diABZI and COVA-i (diABZI + COVA-i) and a conjugate of diABZI and COVA-i (diABZI-COVA-i) were added to the medium, with the corresponding solvent PBS as a negative control. Incubating for 4 hours at 37 ℃, collecting cells, and detecting the expression quantity of the cytokines IFN-beta 1 and CXCL-10 by quantitative PCR.

The results are shown as a in figure 3, from which it can be seen that IFN- β 1 and CXCL-10 expression levels show that both conjugated and unconjugated dianzis exhibit comparable levels of activation of BMDC cells.

DiABZI-COVA-i cross presentation experiment

The basic operation is as follows: BMDC cells were incubated with different combinations of polypeptides at 37 ℃ and antigen presentation and expression levels of mature markers were assessed after 8 hours by flow assay to assess the cross-presentation of antigens by BMDCs.

The method comprises the following specific steps: selecting BMDC cells according to 5 × 10⁵The wells were placed in 12-well plates and after 24 hours medium was added CoVA-i, DiaBZI + COVA-i and DiaBZI-COVA-i to final concentration of 0.5uM, with the corresponding solvent PBS as negative control. Incubate at 37 ℃ for 8 hours, collect cells, wash twice with PBS solution (containing volume fraction 1% FBS), use antibodies CD86 and H2-K^b(SIINFEKI) was stained on ice for 30 minutes, washed twice with PBS solution (containing volume fraction 1% FBS), fixed on 4% w/v paraformaldehyde ice for 20 minutes, and subjected to flow cytometry.

The results are shown in FIG. 3B, from which it can be seen that the level of cross-presentation by BMDC is significantly increased after coupling of COVA-i to DiabZI by flow analysis.

4. Targeted endoplasmic reticulum validation

The basic operation is as follows: adding fluorescent polypeptides with different combinations into DC cells transfected with endoplasmic reticulum tag plasmids, incubating at 37 ℃ for 2 hours, removing the polypeptides, continuing to incubate for 2 hours, fixing the cells, staining cell nuclei, and observing by a confocal microscope.

The method comprises the following specific steps: the endoplasmic reticulum tag plasmid Dsred is placed inElectroporation was carried out in the DC cell line JAWS ll cells according to 1X 10⁵The wells were mounted in 12-well plates containing slides and after 24 hours ova (fitc), diABZ + ova (fitc) and diABZI-ova (fitc) were added to the medium to a final concentration of 5 μ M, with the corresponding solvent PBS as negative control. After incubation for 2 hours at 37 ℃, removing the polypeptide, replacing with a new culture medium and continuing incubation for 2 hours, washing twice with PBS, fixing for 10 minutes at room temperature by 4% w/v paraformaldehyde, staining cell nuclei, and performing ultra-high resolution laser confocal microscopic observation.

The results are shown in FIG. 4. As can be seen in fig. 4, dibizi was able to successfully target the conjugated polypeptide containing the FITC fluorophore to the vicinity of the er matrix. As can be seen from the figure, the fluorescence signals of the OVA (FITC) group and the DiaBZI + OVA (FITC) group are relatively diffuse and flood the whole cells, while relatively concentrated signal spots appear in the DiaBZI-OVA (FITC) group and are highly coincident with the position of the Golgi fluorescent protein of endoplasmic reticulum, which indicates that the DiaBZI can mediate OVA (FITC) to target the endoplasmic reticulum and the Golgi apparatus.

CD8+ T cell expansion experiments

(1) Preparation of CD8+ T cells

Spleens of 7-week-old OT-1 mice (platform model animals, purchased from the center of Guangdong province animals) were collected, dispersed into single cells through a 40 μm cell sieve, erythrocytes were removed with an ACK lysate, and CD8+ T cells were sorted using magnetic beads.

(2) CD8+ T cell expansion experiments

The basic operation is as follows: the selected BMDC cells are incubated with different combinations of polypeptides at 37 ℃, the culture medium is replaced after 12 hours, the BMDC cells and the T cells are co-incubated with CD8+ T cells from O-T1 for 72 hours according to the ratio of 1: 20, and the expansion condition of the CD8+ T cells is analyzed by flow cytometry.

The method comprises the following specific steps: selecting BMDC according to 2 × 10⁴The wells were placed in 12-well plates and after 24 hours medium was added to final concentrations of 0.05 μ M of CoVA-i, DiaBZI + COVA-i and DiaBZI-COVA-i, with the corresponding solvent PBS as negative control. Incubating at 37 deg.C for 12 hr, removing supernatant medium, washing with PBS 1-2 times, adding 4 × 10⁵CFSE (hydroxyfluorescein acetoacetate) dye (5. mu.M, 20 min staining) and CD8+ T cells(ratio of DC cells to T cells 1: 20), incubating at 37 ℃ for 72 hours, collecting CD8+ T cells, staining the cells, fixing the cells with 4% w/v paraformaldehyde, and detecting by a flow cytometer.

As shown in FIG. 5, the co-incubation of BMDC cells treated with DiabZI-COVA-i with O-T1 mouse-derived CD8+ T cells significantly enhanced the amplification effect of the latter. The abscissa a in fig. 5 represents the fluorescence intensity of the CFSE dye, and the weaker the fluorescence, the more the generation number of the amplification, the more the amplification effect of CD8+ T cells is evident, and the ordinate shows the CD8+ T positive cell population and the value of the amplification, respectively. The expanded number of total CD8+ T cells is shown in B in fig. 5.

Claims

1. An endoplasmic reticulum Golgi targeting small molecule, the structural formula of which is shown as the following formula (I):

2. an endoplasmic reticulum Golgi targeting conjugate, the structural formula of which is shown in the following formula (II):

wherein R is₁Is that

R₂Is that

R₃Is an antigen.

3. The Nephron golgi targeted conjugate of claim 2, wherein: the antigen is a polypeptide antigen or a protein antigen.

4. The Nerium Golgi targeted conjugate according to any one of claims 2 or 3, wherein: r₃Is an antigen containing cysteine at the N-terminal.

5. Use of the Neigold matrix-targeting conjugate of any one of claims 2 to 4 for the preparation of a STING protein agonist.

6. Use of an Nerium Golgi targeted conjugate according to any one of claims 2 to 4 for the preparation of a medicament for increasing the level of cross-antigen presentation.

7. Use of the reticulum endoplasmic reticulum Golgi targeting conjugate of any one of claims 2 to 4 for the preparation of an immunotherapeutic drug.

8. Use of an endoplasmic reticulum Golgi targeting conjugate according to any one of claims 2 to 4 in the manufacture of a medicament for enhancing antigen targeting to the endoplasmic reticulum.

9. Use of an Neigold matrix-targeting conjugate according to any one of claims 2 to 4 in the manufacture of a medicament for inducing a CD8+ T immune response.

10. The use of the Nerium Golgi targeted small molecule of claim 1 for preparing a STING protein agonist, or for preparing a medicament for increasing the level of antigen cross-presentation, or for preparing a medicament for enhancing antigen targeting to the Nerium, or for preparing a medicament for inducing CD8+ T immune response, or for preparing an immunotherapeutic medicament.